Study of biological functions and molecular mechanisms of programmed death-ligand 1 protein in acute myeloblastic leukemia

Abstract

Acute Myeloblastic Leukemia (AML) is a type of acute leukemia that caused by clonal disorder leading to the abnormal of myeloid proliferation and differentiation. Nowadays, chemotherapy has been used as a standard treatment for AML, however it has drawbacks such serious side effects and drug resistance. Within 2–3 years of receiving initial therapy, 50% of all AML patients who achieved remission are vulnerable to relapse. To prevent the harmful effects of chemotherapy, new therapeutic options are required. As immune checkpoint inhibitors (ICIs) continue to advance, increasing evidence have demonstrated that anti-PD-1/PD-L1 immunotherapy is an effective treatment against cancers. The co-inhibitory ligand known as programmed death ligand-1 (PD-L1) contributes to T-cell exhaustion by interacting with programmed death-1 (PD-1) receptor. Therefore, numerous studies have focused on communication between cancer cells and T-cells. The previous study indicated that PD-L1 not only mediates tumor-immune cell communication, but also exerts independent intracellular functions in cancer cells themselves. There are few studies focus the role of PD-L1 in the mechanism underlying AML development, and the PD-L1 associated intrinsic signaling network in the leukemic cells is not well investigated in leukemia. Therefore, to fully understand the role of PD-L1 in the biological function of leukemic cells, the data for AML patients were extracted from TCGA and GTEx databases. The bioinformatic data revealed that PD-L1 was upregulated in AML patients when compared to the normal group and the high expression of PD-L1 gene was significantly associated with poor overall survival. The downstream signaling pathways of PD-L1 were identified via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. It was found that the key downstream pathways of PD-L1 were identified to be the ECM-receptor interaction and the PI3K-AKT signaling pathway. Then, the key PD-L1 related genes were selected by Weighted gene co-expression network analysis (WGCNA), Maximum Correntropy Criterion (MCC) algorithm, and Molecular Complex Detection (MCODE). By all three analyses, eight genes (ITGA2B, ITGB3, COL6A5, COL6A6, PF4, NMU, AGTR1, F2RL3) were significantly associated to PD-L1. For in vitro study, leukemic cell lines, KG-1a and EoL-1, which represent positive and negative PD-L1 cell lines respectively, were used to investigate the effect of PD-L1 expression on biological functions of AML. By CCK-8 assay, the proliferation rate of KG-1a with small interfering RNA against PD-L1 (siPDL1) groups was significantly lower than that of transfected negative (siNC) group both at 48 and 72 h (P < 0.05). Upon PD-L1 overexpression, the proliferation rate of EoL-1 with PD-L1 overexpressing group was significantly higher than that of the EoL-1 cells with vector control group (P < 0.05). For cell apoptosis, the percentages of apoptotic cells were 11.46 and 12.08% in siPDL1#1 and siPDL1#2 group, respectively, compared with 7.69 % in siNC group (P < 0.001) in KG-1a cell line. Furthermore, cell cycle analysis revealed that the percentage of KG-1a cells in G2/M phase was 19.22% in siPDL1#2 group and 4.35% in siNC group. To clarify the mechanism by which PD-L1 regulates cell proliferation and cell apoptosis, SDS-PAGE and Western blotting were carried out. The results showed that the expression of caspase-7 and cleaved caspase-3 proteins were increased following PD-L1 knockdown in KG-1a cells, but that of PI3K and p-AKT were decreased. In contrast，KG-1a and EoL-1 cells showed increased PI3K, AKT, and p-AKT expressions after overexpressing PD-L1 protein. In the PD-L1-overexpressing KG-1a cells, the AKT inhibitor MK-2206 completely inhibited PD-L1-promoted cell proliferation. Furthermore, PD-L1 overexpression promoted the expression of the PAR4 protein in KG-1a cells. While, silencing the PAR4 gene inhibited PD-L1 expression, which in turn prevented the up-regulation of KG-1a cell proliferation. The flow cytometric analysis revealed that there were 8.5 and 14.3% apoptotic cells in siNC-PDL1 and siPAR4-PDL1 groups, respectively when compared to 12.2% apoptotic cells in siNC-NC group. Knockdown of PAR4 gene expression can reverse the effects of PD-L1 overexpression in KG-1a cell cycle. Following PAR4 knockdown in KG-1a cells, the expressions of PI3K and p-AKT had no changed, whereas MK-2206 (AKT inhibitor) was able to down-regulate PAR4 gene expression. These findings could be proposed the mechanism that PD-L1 regulated biological behaviors in leukemia cell lines including cell proliferation, cell cycle arrest, and cell apoptosis mediated by PI3K-AKT-PAR4 signaling pathway. Taken together, the current study reveals the major regulatory network and downstream targets of PD-L1 in AML, elucidating the underlying mechanism of anti-PD-1/PD-L1 immunotherapy and paving the way to further clinical applications of ICIs in AML.